Project Summary/Abstract Genomic technologies have revealed extensive transcription through ?non-coding? regions of genomes to yield regulatory RNAs that control development and physiology. A host of cellular constituents implicated in regulating RNAs function in broadly expressed multimeric complexes. Many questions remain unanswered regarding how these complexes control differentiation, proliferation and survival, and how they function in cell type-specific contexts, e.g. in diverse cells of the hematopoietic system. We discovered that the RNA- regulatory exosome complex (exosome) opposes primary erythroid cell maturation, and the erythroid transcription factor GATA-1 represses genes encoding exosome subunits. Downregulating exosome subunits abrogates intra-complex protein-protein interactions and promotes erythroid maturation. Our progress revealed the exosome confers expression of the vital receptor tyrosine kinase c-Kit that mediates Stem Cell Factor (SCF) pro-proliferation signaling in erythroid precursors, while opposing pro-differentiation erythropoietin (Epo) signals. These results establish a paradigm in which an RNA-regulatory complex orchestrates a developmental signaling transition to balance proliferation and differentiation. This paradigm has considerable importance for understanding mechanisms governing erythrocyte genesis in physiological and pathological states and provides a foundation for devising strategies to control this process independent of Epo-dependent interventions. In one aim, as specified by SHINE II, we propose mechanistic/biological analyses to elucidate how the exosome regulates c-Kit expression/function in primary mouse and human erythroid cells. In Aim 1, we will elucidate how the exosome confers SCF/c-Kit signaling. Exosome dismantling decreases Kit mRNA and primary transcript levels, the opposite response predicted from exosome function to degrade RNAs. As the exosome occupies insulators and promoters, is implicated in superenhancer function and chromatin modification, and suppresses promoter upstream transcripts that regulate genes, we predict it directly confers Kit transcription (Model 1). Since the exosome degrades regulatory RNAs, exosome dismantling might elevate regulatory RNA(s) that act in trans to repress Kit (Model 2). These equally important mechanisms represent a new dimension on how cells mount Stem Cell Factor (SCF) signaling. In Aim 1a, we will test the hypothesis that direct exosome function at Kit regulates transcription. In Aim 1b, we will test whether exosome dismantling selectively or broadly expels Pol II and/or its functionally distinct isoforms. In Aim 1c, we will test whether the mechanism operates in primary human erythroid cells, which is supported by compelling initial data. These studies will unravel a mechanism governing acquisition of SCF/c-Kit signaling and how the exosome controls erythroid maturation. Given the crucial c-Kit functions in normal and malignant hematology, regenerative biology and more broadly, mechanistic analyses of this innovative paradigm will yield findings of high fundamental and translational significance.